The atrial natriuretic peptide (ANP) receptor is involved in renal and cardiac functions and regulates blood pressure. The receptor is stimulated by natriuretic peptide binding which potently increases natriuresis and diuresis in the kidney. ANP has shown to be protective against nephrotoxicity caused by immuno-suppresive drugs and also has clinical benefits for congestive heart failure patients. The ANP receptor belongs to the family of membrane bound guanylyl cyclases and contains an extracellular ligand binding domain, a transmembrane helix, a kinase-like domain, a coiled-coil, and guanylyl cyclase domain. Extracellular ANP binding to this receptor leads to the intracellular production of the second messenger cGMP. Our long term goal is to determine the molecular signal transduction events of the ANP receptor using protein crystallography. We have previously determined the 2.0 Angstrom crystal structure of the extracellular ligand binding domain. This pilot and feasibility study now focuses on obtaining diffracting crystals of the full length and intracellular domains of the ANP receptor. Specific Aim 1 is to crystallize the full length ANP receptor. Our two collaborators have worked out the purification of the full length ANP receptor from bovine adrenal gland to homogeneity and have shown the feasibility of obtaining and partially purifying ANP receptor from over-expressing HEK293 cells. The ANP receptor can readily be solubilized in Triton X-100 and efficient purification protocols involve affinity chromatography steps involving ANP-agarose or antibody-based methods. We plan to scale up the protein purification efforts and carry out extensive (co)-crystallization efforts. The ANP receptor is arguably an ideal candidate for crystallizing a single transmembrane helix receptor due to its dimeric nature and purification options. Specific Aim 2 is to crystallize the intracellular domains of the ANP receptor. We have developed a suitable expression system for the intracellular domains using a baculovirus/Sf9 system and have worked out a purification scheme to obtain catalytically active ANP receptor intracellular domain protein. We will crystallize the intracellular domains in the presence and absence of ATP and GTP analogs. Our long term structural studies on the molecular mechanism of ANP receptor signaling could enhance the development of renal and cardiovascular drugs. [unreadable] [unreadable] [unreadable]